Increased Frequency and Vasculogenic Potential of Endothelial Colony-Forming Cells in Patients with Kaposi’s Sarcoma  Francesca Calcaterra, Lucia Brambilla, Elena Colombo, Athanasia Tourlaki, Stefano Veraldi, Claudia Carenza, Domenico Mavilio, Silvia Della Bella  Journal of Investigative Dermatology  Volume 137, Issue 7, Pages 1533-1540 (July 2017) DOI: 10.1016/j.jid.2017.02.979 Copyright © 2017 The Authors Terms and Conditions

Figure 1 The frequency of circulating ECFCs is highly increased in patients with cKS. (a) The number of ECFC colonies isolated from the peripheral blood was much higher in cKS patients (n = 86) than healthy control subjects (n = 83). (b) ECFC colonies isolated from cKS patients appeared some days earlier than those from healthy donors. (c) Within the group of cKS patients, the increased frequency of ECFC colonies was more pronounced in patients with rapidly evolving (cKS B, n = 52) than slowly evolving (cKS A, n = 15) disease. Data are presented as mean ± standard error of the mean. P-values calculated by the Mann-Whitney U test. cKS, classic Kaposi’s sarcoma; ECFC, endothelial colony-forming cell; PBMC, peripheral blood mononuclear cell. Journal of Investigative Dermatology 2017 137, 1533-1540DOI: (10.1016/j.jid.2017.02.979) Copyright © 2017 The Authors Terms and Conditions

Figure 2 ECFCs isolated from cKS patients are endowed with higher proliferative and vasculogenic potential. (a) ECFCs from cKS patients (n = 45) displayed at the end of the culture more than 10-fold higher cell yield than ECFCs obtained from healthy donors (n = 45). (b) The higher cell yield of ECFCs from cKS patients was evident from the initial phases of cell culture, because ECFCs obtained from cKS patients gave rise to a higher number of cells than control ECFCs starting from the first week of culture. (c) Within the group of cKS patients, the cell yield of ECFCs tended to be higher in patients with rapidly evolving (cKS B, n = 24) than slowly evolving (cKS A, n = 21) disease. (d) Representative photographs of tubules formed in Matrigel matrix (Corning Inc., Corning, NY) by ECFCs obtained from one healthy donor and one cKS patient (original magnification ×4, scale bar = 500 μm). (e) ECFCs isolated from cKS patients (n = 32) formed a significantly higher number of tubules than those from healthy donors (n = 27). Data are presented as mean ± standard error of the mean. P-values calculated by the Mann-Whitney U test. cKS, classic Kaposi’s sarcoma; ECFC, endothelial colony-forming cell; n.s., not significant. Journal of Investigative Dermatology 2017 137, 1533-1540DOI: (10.1016/j.jid.2017.02.979) Copyright © 2017 The Authors Terms and Conditions

Figure 3 ECFCs isolated from cKS patients produce higher levels of IL-6. (a) Representative curves from one healthy donor and one cKS patient are presented, showing that the levels of IL-6 in ECFC supernatants increase progressively along with culture progression. (b) ECFC cultures obtained from cKS patients (n = 34) reached higher levels of IL-6 than ECFCs obtained from healthy donors (n = 34). (c) Within the group of cKS patients, the levels of IL-6 tended to be higher in patients with rapidly evolving (cKS B, n = 17) than slowly evolving (cKS A, n = 17) disease (P = 0.24). Data presented as mean ± standard error of the mean. P-values calculated by the Mann-Whitney U test. cKS, classic Kaposi’s sarcoma; ECFC, endothelial colony-forming cell; n.s., not significant. Journal of Investigative Dermatology 2017 137, 1533-1540DOI: (10.1016/j.jid.2017.02.979) Copyright © 2017 The Authors Terms and Conditions

Figure 4 ECFCs isolated from cKS patients are latently infected by HHV8. Photographs of LANA-1 staining observed in (a) the BCBL-1 cell line latently infected by HHV8, used as positive control (original magnification ×120), (b) ECFCs obtained from one representative of six healthy HHV8-seronegative donors (original magnification ×120), and (c) ECFCs obtained from three representatives of six different cKS patients, showing positive LANA-1 expression in a small proportion of cells in each colony (original magnification ×120). Scale bars = 10 μm. Insets for cKS patients shown at higher magnification (original magnification ×240). Arrows indicate the localization of LANA-1 staining in ECFCs. cKS, classic Kaposi’s sarcoma; ECFC, endothelial colony-forming cell; HHV8, human herpesvirus-8. Journal of Investigative Dermatology 2017 137, 1533-1540DOI: (10.1016/j.jid.2017.02.979) Copyright © 2017 The Authors Terms and Conditions